(i) Field of the Invention
The present invention relates to genetically modified plant cells and plants in which the genetic modification leads to the synthesis of amylopectin starch with novel properties. Furthermore, the present invention relates to methods for the production of such plant cells and plants. The present invention also relates to starches produced by the plant cells and plants of the invention and methods for the production of these starches and derivatised starches.
(ii) Description of the Related Art
In view of the increasing significance that is currently being attributed to vegetable ingredients as sources of renewable raw materials, one of the objectives of bioengineering research is concerned with the adaptation of these vegetable raw materials to the requirements of the processing industry. In order to be able to utilise renewable raw materials in as many areas as possible it is additionally necessary to identify a wide variety of substances.
Starch is a complex mixture of polysaccharides that are composed of chemically unique base components, glucose molecules. However, polysaccharide molecules differ in respect of the degree of polymerisation and branching, which influences the physico-chemical properties of starch. A differentiation is made between amylose starch, i.e. starch that is comprised mainly of amylose, and amylopectin starch, i.e. starch that is comprised mainly of amylopectin.
For a considerable time amylose was regarded as a linear polymer, consisting of α-1,4-glycosidically linked α-D-glucose monomers. However, more recent studies have proven the presence of α-1,6-glycosidic branching points (approx. 0.1%) (Hizukuri and Takagi, 1984, Carbohydr. Res. 134, 1-10; Takeda et al., 1984, Carbohydr. Res. 132, 83-92).
Amylopectin consists of a complex mixture of variably branched glucose chains. Unlike amylose, amylopectin is more highly branched. Side chains are linked to the primary chain, consisting of α-1,4-glycosidically linked α-D-glucose monomers, by way of α-1,6-glycosidic bonds. According to textbook data (Voet and Voet, Biochemistry, John Wiley & Sons, 1990), the α-1,6 branches occur on average every 24 to 30 glucose moieties. This corresponds to a degree of branching of approx. 3%-4%. The data regarding the degree of branching are variable and dependent on the origin of the respective starch (e.g. plant species, plant variety, etc.). In plants that are typically used for industrial starch production such as, for example, maize, wheat or potato, the starch synthesised is comprised of approx. 20%-30% amylose starch and approx. 70%-80% amylopectin starch. Another fundamental difference between amylose and amylopectin lies in the molecular weight. amylose, depending on the origin of the starch, has a molecular weight of 5×105-106 Da, in the case of amylopectin it lies between 107 and 108 Da. The two macromolecules can be differentiated on the basis of their molecular weight and their differing physico-chemical properties, which can be seen most readily in their different iodine binding properties.
In addition to the amylose/amylopectin ratio and the phosphate content the functional characteristics of the starch are strongly influenced by the molecular weight, the side chain distribution pattern, the ion content, the lipid and protein content, the average starch granule size and the starch granule morphology. Examples of important functional characteristics are the solubility, the retrogradation behaviour, the water binding capability, the film formation properties, the viscosity, the gelatinisation properties, the freeze/thaw stability, the acid stability, the gel solidity, etc.
The gelatinisation properties, which include the end viscosity, can be determined by the person skilled in the art with various methods. Depending on the method used, absolute values in particular, but also relative values, can differ for the same starch sample. A quick and efficient method for the analysis of gelatinisation properties is the RVA analysis. Depending on the parameters selected and the temperature profile during the RVA analysis, different RVA profiles are obtained for the same sample. It should be noted that in the following quoted documents, which explain the state of the art, different profiles are sometimes described for determining the agglutination properties.
It is known that plants can be genetically modified in such a way that they produce starch that can be differentiated on the basis of physico-chemical parameters from the starch that is manufactured by corresponding plants that have not been genetically modified. A review of various plant species that exhibit a reduction in enzymes involved in the starch biosynthesis has been described by Kossmann and Lloyd (2000, Critical Reviews in Plant Sciences 19(3), 171-126).
In conjunction with the present invention the following state of the art is of interest. Plants have hitherto been described in which the activity of the starch granule-bound starch synthase GBSSI (“Granule-Bound Starch Synthase”) is reduced (Shure et al., 1983, Cell 35, 225-233; Hovenkamp-Hermelink et al., 1987, Theoretical and Applied Genetics 75, 217-221; Visser et al., 1991, Mol. Gen. Genet. 225, 289-296; Hergersberg, 1988, Dissertation, Universität Köln; WO 92/11376). The GBBSI is involved in the formation of amylose. Inhibition of the GBSSI activity leads to a synthesis of starch that is comprised almost exclusively of amylopectin. The corresponding GBSSI gene in the maize plant is known by the term “waxy”.
Furthermore, plants have been described in which the activity of soluble starch synthase SSIII is reduced (Abel et al., 1996, The Plant Journal 10(6), 981-991; Lloyd et al., 1999, Biochemical Journal 338, 515-521; WO 00/08184; WO 96/15248; EP-A 0779363). In comparison with starch isolated from corresponding wild type plants, starch from such plants exhibits a relative shift of the side chains of amylopectin from longer chains to shorter chains (Lloyd et al., 1999, Biochemical Journal 338, 515-521), an increased phosphate content, no change in the amylose content (Abel et al., 1996, The Plant Journal 10(6), 9891-9991) and a reduced end viscosity in the RVA analysis (Abel, 1995, Dissertation, Freie Universität Berlin).
Furthermore, plants have been described, in with the activity of the branching enzyme BEI is reduced (Kossmann et al., 1991, Mol. Gen. Genet. 230, 39-44; Safford et al., 1998, Carbohydrate Polymers 35, 155-168; WO 92/14827; WO 95/26407). Safford et al. (1998, supra) describe that corresponding potato plants produce a starch with an amylose/amylopectin ratio that is essentially unchanged. Nor does the degree of branching of the amylopectin differ significantly from that of starch isolated from wild type potato plants. The starch-bound phosphate content is comparably increased, however, which presumably leads to the various gelatinisation properties observed and the altered viscosity of the starch isolated from corresponding mutants as compared to starch isolated from wild type potato plants.
Plants are described in WO 01/19975, in which both the GBSSI and the SSII and/or SSIII activity are reduced. Starch from potato plants with reduced GBSSI, SSII and SSIII activity exhibits a lower amylose content, changed swelling properties and gelatinisation properties, and an increased freeze/thaw stability in comparison to starch from wild type potato plants.
Plants are described in WO 01/12782, in which both the GBSSI and the BEI activity are reduced. In comparison to starch from wild type plants, starch from these plants exhibits a reduced amylose content, and in comparison to starch from plants of the waxy phenotype an increased phosphate content and/or lowered gelatinisation temperature. Plants are described in WO 00/08184, in which both the SSIII and the BEI activity are reduced. In comparison to starch from wild type plants starch from such plants exhibits an increased phosphate content.